Engineered telomeres in transgenic Xenopus laevis

The expanding roles of telomeres in epigenetic gene regulation, nuclear organization, and human disease have necessitated the establishment of model organisms in which to study telomere function under normal developmental conditions. We present an efficient system for generating numerous vertebrate animals containing engineered telomeres using a Xenopus laevis transgenesis technique. Our results indicate Xenopus zygotes efficiently recognize telomeric repeats at chromosome break points and form telomeric complexes thus generating a new telomere. The resulting transgenic animals progress through normal development and successfully metamorphose into froglets despite the chromosome breakage. Overall, this presents an efficient mechanism for generating engineered telomeres in a vertebrate system and provides an opportunity to investigate epigenetic aspects of telomere function during normal vertebrate development.     

Developmental immunohistology of melanotrophs in Xenopus laevis tadpoles

Summary The adenohypophysial primordium of Xenopus laevis tadpoles at stages 33/34 to 46 (Nieuwkoop and Faber, 1956) were examined immuno-histologically for -MSH, -MSH and ACTH. -MSH was demonstrated from stage 37/38 onwards, and -MSH from stage 39. No signs of ACTH production were detected. -MSH and -MSH occurred in the same cells. No differences were found in the intensity of immunofluorescence between tadpoles which were kept on a black and a white background. The present study lends no support to the hypothesis concerning the derivation of -MSH from ACTH. The observations made suggest that the morphological formation of the pars intermedia is accomplished during stages 37/38 to 39. Acknowledgement. The authors express warm thanks to Dr. M.P. Dubois (Laboratoire de Physiologie de la Reproduction, INRA, Nouzilly, France), who prepared and verified the antibodies. Grants from Swedish Natural Science Research Council and Landshovding Per Westlings minnesfond, Lund, Sweden are gratefully acknowledged     

LHRH-like system in the brain of Xenopus laevis daud

Summary Rabbit antiserum to synthetic LHRH was used with the immunofluorescence technique to identify the LHRH-secreting neurons and their axonal pathways in the brain of Xenopus laevis. Three groups of immunoreactive neurons were identified: the first, in the telencephalon, is a paired group of cells scattered near the two telencephalic ventricles; the second group lies near the preoptic recess; the third group occurs in the ventral wall of the infundibulum. Two principal neuronal pathways were observed: Fibres originating from the dorsally located telencephalic neurons converge on the cephalic median plane where they form a single bundle behind the telencephalic furrow. This bundle descends towards the anterior border of the preoptic recess where it divides into two nerve bundles which pass on either side of the preoptic recess, run above the optic chiasma then cross the infundibular floor and finally terminate in the median eminence. The second pathway is more direct. The more ventrally located telencephalic LHRH cells give rise to this second pathway. Their axons converge with the other LHRH fibres near the lateral border of the preoptic recess. Most of the LHRH nerve fibres terminate in the median eminence although some terminate near the paired pars tuberalis. No reaction was observed after the use of antiserum absorbed with synthetic antigen.Equipe de Recherche associée C.N.R.S. n 492. This work was financed by the D.G.R.S.T., Contract n 7470046     

Ultrastructural observations of the tunica muscularis in the small intestine of Xenopus laevis, with special reference to the interstitial cells of Cajal

The distribution and ultrastructure of the interstitial cells of Cajal (ICC) has been examined in the small intestine of the frog Xenopus laevis, as the physiological significance of these cells remains obscure in amphibians and other lower vertebrates. The present study has revealed the existence of a special type of interstitial cell in the tunica muscularis of the small intestine of Xenopus; this cell is characterized by the presence of numerous caveolae, many small mitochondria, and the formation of intercellular connections with the same type of cell. Since these ultrastructural features are shared with mammalian ICC, the cells in the small intestine of Xenopus probably correspond to ICC. These cells also form close contacts with neighboring smooth muscle cells and with nerve varicosities containing accumulations of synaptic vesicles. These cellular networks are likely to be involved in the transmission of nerve impulses to muscle cells, as has been suggested for mammalian tissues. However, true gap junctions have not been detected; they occur neither between the same type of cells nor between the putative ICC and smooth muscle cells. The widespread distribution of ICC or equivalent cells in different groups of vertebrates, together with the conservation of their ultrastructural features, suggests that they differentiated early in vertebrate evolution to play key regulatory roles in gastrointestinal movement.     

Structure of the main ganglioside from the brain of Xenopus laevis

The main component of the ganglioside¹ mixture from the brain of the adult amphibian Xenopus laevis accounts for 35% of the total, as lipid bound sialic acid. This ganglioside has been purified and characterized by thin layer chromatography, partial and exhaustive enzymatic hydrolysis with sialidase, TLC-overlay procedures with anti-Gg₄Cer and anti-Neu5Ac6GalNAc specific monoclonal antibodies and mass spectrometry. All together the results suggest the following structure:Neu5Ac8Neu5Ac3Gal3(Neu5Ac8Neu5Ac6)GalNAc4Gal4Glc1Ceror, IV³--Neu5Ac₂,III⁶--Neu5Ac₂-Gg₄Cer.     

Aroclor 1254 impairs the hearing ability of Xenopus laevis

In this study we assessed the effects of chronic, dietary exposure of Aroclor 1254 (A1254) on the hearing of Xenopus frogs. We used the auditory brainstem response (ABR) to assay changes in hearing physiology; ABR thresholds, as well as latency-intensity and amplitude-intensity profiles of the initial positive (P1) and negative (N1) peaks were measured. Two groups of animals that received 50 ppm and 100 ppm of A1254 in their diet from 5 days post-fertilization through metamorphosis were compared to a control group that received untreated chow. The results showed significant threshold elevations in the 3–4 kHz range and significantly delayed peak latencies and reduced amplitudes at these frequencies in A1254 treated animals as compared to control animals. These findings indicate that A1254 selectively damages the high-frequency sensorineural hearing system associated with the basilar papilla of frogs. This preferential damage may be related to inherent differences in the vulnerability of the basilar versus amphibian papilla in the frog. The overall results of this study are also consistent with the reported A1254-induced auditory deficits in mammals indicating that the basilar papilla of the Xenopus frog may serve as an effective model for studying the effects of A1254 on the auditory system.     

Fission yeast tmsl protein abrogates normal development in Xenopus laevis embryos

Recently we cloned tms1 (a putative dehydrogenase) by complementation of a human tumour-derived mutant p53 induced growth arrest in fission yeast. Microinjection of purified tmsl protein into Xenopus laevis embryos abrogated normal embryo development by causing cleavage retardation or cleavage arrest of injected blastomeres in a concentration dependant manner, whereas injection of specific affinity purified tms1 antiserum showed no significant morphological defects. Microinjection of tms1 protein together with affinity purified tms1 antibody resulted in a significantly reduced number of cleavage arrested embryos.     

Changing complexity of endogenous lectin activities during juvenile development of Xenopus laevis

Galactoside-binding lectin has been isolated from whole Xenopus laevis embryos and tadpoles at four development stages: st. 24–26, 32, 41 and 47. The main lectin activity at st. 24–26 is -galactoside specific, producing a 34/35.5K doublet on SDS-PAGE. Later in development, lectin activities specific for a wide range of other sugars appear concommitant with the detection of a number of new protein bands on SDS-PAGE gels. The greatest variety of new lectin activities exists at st. 32 when lectins specific for all of the main sugar families found in nature are detected. After this stage and up to st. 47 (the beginning of metamorphosis), fewer different lectin activities are again detected. The results suggest that a complex, developmentally regulated battery of different lectins are present during early Xenopus development, perhaps with stage-specific roles to play in the control of tissue morphogenesis.     

Ultrastructural observations on the germ line of Xenopus laevis

Summary The development of the male germ line in Xenopus laevis has been examined by electron microscopy. Findings have been compared to the parallel process in the female. Three structures unique to the germ line were found in both male and female cells: a fibrillar nuclear region free of DNA; largely proteinaceous masses of nuage material; and a chromatoid body. Germ plasm bodies of the egg and early embryo appear to represent a form of nuage material. The finding of a structure which can be identified as a chromatoid body in the female germ line is unique, as is its presence in sexually undifferentiated primordial germ cells. The chromatoid body in Xenopus, unlike that in mammals, does not persist in the spermatozoon. Instead, it dissociates into a series of coated vesicles during spermatogenesis. The chromosomal ultrastructure of meiotic prophase stages in Xenopus is similar in both sexes until diplotene, when male bivalents condense and enter meiotic metaphase instead of entering the extended lampbrush stage characteristic of the female. The multiple nucleoli present in gonia are lost at the onset of meiotic prophase, but no obvious mechanism for this process was observed.The author would like to thank Drs. Joseph Gall and Bernard Tandler for their helpful suggestions during the course of this investigation. The author is a postdoctoral fellow of the National Institutes of Health, U.S.A. This research was supported by N.I.H. Grants 51823 and 12427.     

The area octavo-lateralis in Xenopus laevis

Summary Central projections of afferents from the lateral line nerves and from the individual branches of the VIIIth cranial nerve in Xenopus laevis and Xenopus mülleri were studied by the application of HRP to the cut end of the nerves.Upon entering the rhombencephalon, the lateral line afferents form a longitudinal fascicle of ascending and descending branches in the ventro-lateral part of the lateral line neuropile. The fascicle exhibits a topographic organization, that is not reflected in the terminal field of the side branches. The terminal field can be subdivided into a rostral, a medial and a caudal part, each of which shows specific branching and terminal pattern of the lateral line afferents. These different patterns within the terminal field are interpreted as the reflection of functional subdivisions of the lateral line area. The study did not reveal a simple topographic relationship between peripheral neuromasts and their central projections.Two nuclei of the alar plate with significant lateral line input were delineated: the lateral line nucleus (LLN) and the medial part of the anterior nucleus (AN). An additional cell group, the intermediate nucleus (IN), is a zone of lateral line and eighth nerve overlap, although such zones also exist within the ventral part of the LLN and the dorsal part of the caudal nucleus (CN). Six nuclei which receive significant VIIIth nerve input are recognized: the cerebellar nucleus (CbN), the lateral part of the anterior nucleus, the dorsal medullary nucleus (DMN), the lateral octavus nucleus (LON), the medial vestibular nucleus (MVN) and the caudal nucleus (CN).All inner ear organs have more than one projection field. All organs project to the dorsal part of the LON and the lateral part of the AN. Lagena, amphibian papilla and basilar papilla project to separate regions of the dorsal medullary nucleus (DMN). There is evidence for a topographic relation between the hair cells of the amphibian papilla (AP) and the central projections of AP fibers. The sacculus projects extensively to a region between the DMN and the LON. Fibers from the sacculus and the lagena project directly to the superior olive. Fibers from the utriculus and the three crista organs terminate predominantly in the medial vestibular nucleus (MVN) and in the adjacent parts of the reticular formation, and their terminal structures appear to be organotopically organised. Octavus fiber projections to the cerebellum and to the spinal cord are also described.     

In-cell NMR spectroscopy of proteins inside Xenopus laevis oocytes

In-cell NMR is an application of solution NMR that enables the investigation of protein conformations inside living cells. We have measured in-cell NMR spectra in oocytes from the African clawed frog Xenopus laevis. ¹⁵N-labeled ubiquitin, its derivatives and calmodulin were injected into Xenopus oocytes and two-dimensional ¹H–¹⁵N correlation spectra of the proteins were obtained. While the spectrum of wild-type ubiquitin in oocytes had rather fewer cross-peaks compared to its in vitro spectrum, ubiquitin derivatives that are presumably unable to bind to ubiquitin-interacting proteins gave a markedly larger number of cross-peaks. This observation suggests that protein–protein interactions between ubiquitin and ubiquitin-interacting proteins may cause NMR signal broadening, and hence spoil the quality of the in-cell HSQC spectra. In addition, we observed the maturation of ubiquitin precursor derivative in living oocytes using the in-cell NMR technique. This process was partly inhibited by pre-addition of ubiquitin aldehyde, a specific inhibitor for ubiquitin C-terminal hydrolase (UCH). Our work demonstrates the potential usefulness of in-cell NMR with Xenopus oocytes for the investigation of protein conformations and functions under intracellular environmental conditions.Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at .     

Nerve-independent DNA synthesis and mitosis in regenerating hindlimbs of larval Xenopus laevis

Summary Xenopus laevis larvae at stage 52–53 (according to Nieuwkoop and Faber 1956) were subjected to amputation of both limbs at the thigh level as well as to repeated denervations of the right limb. Results obtained in larvae sacrificed during wound healing (1 after amputation), blastema formation (3 days) and blastema growth (5 and 7 days) showed that denervated right limbs have undergone the same histological modifications observed in innervated left limbs and have formed a regeneration blastema consisting of mesenchymal cells with a pattern of DNA synthesis and mitosis very similar to that in presence of nerves. Also, the patterns of cellular density in regenerating right and left limbs were very similar. On the whole, the data here reported show a highly remarkable degree of nerve-independence for regeneration in hindlimbs of larval Xenopus laevis at stage 52–53 and lend some substance to the hypothesis that, in early limbs, there would exist trophic factors capable of replacing those released by nerves, promoting DNA synthesis and mitosis in blastemal cells. Offprint requests to: S. Filoni     

The distribution of nucleoplasmin in early development and organogenesis of Xenopus laevis

Summary The fate of the germinal vesicle-derived protein, nucleoplasmin, was followed in embryos and tadpoles of Xenopus using monoclonal antibodies and indirect immunofluorescent staining. Nucleoplasmin was found in all nuclei up to feeding tadpole stages. Thereafter its level decreased in all nuclei. It was not detected in nuclei of advanced tadpoles or of adults. Contrasting with another protein, N₁, that was previously monitored in the nuclei of dividing gonia of both sexes, nucleoplasmin was only detected in the nuclei of ovarian oocytes starting at diplotene. Traces of nucleoplasmin have also been found in a rapidly-dividing fibroblastic cell-line by immunohistology and protein blotting.     

The relationship of innervation and differentiation to regenerative capacity in the reamputated hindlimb of larval Xenopus laevis

Regenerated hindlimbs of larval Xenopus laevis were reamputated at critical larval stages and levels, viz when amputation of the control limb at the same larval stage and level is followed by reduced regeneration. Reamputations were performed at the level of (1) the original plane of amputation, (2) the early regenerate (cone/palette stage), (3) the late regenerate (digit stage). Reamputation increased both the percentage rate of regeneration and the morphological complexity of the regenerates in all experimental series. Cell counts in lateral motor columns and spinal ganglia innervating the hindlimb, together with histological observations and mitotic index and labelling index determinations in reamputated and control limbs showed that improved regeneration in the reamputated limb was related to an increase in undifferentiated and proliferating cells in the stump. We did not find any evidence suggesting that renewed regeneration in reamputated anuran limbs results from an increase in innervation, as has previously been hypothesized. We support our conclusions by demonstrating an improvement in regenerationen in the reamputated and denervated hindlimbs.     

Some morphogenetic features of the adenohypophysial primordium of early Xenopus laevis tadpoles

Summary The adenohypophyses of Xenopus laevis tadpoles at developmental stages 20 to 46 (Nieuwkoop and Faber, 1956) were studied. From its first appearance at about stage 20 to 21, the adenohypophysial primordium passes through four morphogenetic phases, each characterized by internal events. The first phase (stages 20 to about 33/34) is characterized by extensive proliferation of the primordium. During the second phase (stages 33/34 to about 37/38), the growth of the primordium is arrested. This arrest coincides with the attainment of secretory function. The primordium is claviform in shape at these stages. The third phase, roughly stage 39, is characterized by a thorough reorganization of the adenohypophysial cells, leading to the formation of the pars distalis and pars intermedia. The shape of the primordium changes, and its volume temporarily increases. The last phase is characterized by the organization of the pars distalis cells into cell cords which possibly demonstrate a functional relation to a specialized region (the hilus) of the adenohypo-physis-brain interspace. Acknowledgements. Grants from the Faculty of Mathematics and Science, University of Lund, the Royal Physiographic Society, Lund, and the Swedish Natural Sience Research Council are gratefully acknowledged     

Crystallins during Xenopus laevis free lens formation

Summary The ontogeny and localization of crystallins during free lens development (i.e. lens development without the optic vesicle) were investigated in Xenopus laevis using the indirect immunofluorescence staining method with an antiserum raised against homologous total lens soluble proteins. Since the developing free lenses pass through stages similar to those of the lenses regenerated from the inner cell layer of the outer cornea following lentectomy in the same species Freeman's classification was used to identify the stages of free lens development. The first appearance of a positive reaction occurred at early stage IV in a number of cells in an area where future lens fibre cells would develop. With further differentiation of the free lens more and more cells in the fibre area started to show a positive reaction and the first positive reaction in the epithelium was observed late in stage V. Histological examination revealed that a fully differentiated free lens and a normally developed lens are similar but that the free lens is smaller.     

Ultrastruktur der spinalen Liquorkontaktneurone beim Krallenfrosch (Xenopus laevis)

Zusammenfassung Zwischen und unter den Ependymzellen des Zentralkanals des Rückenmarkes von Xenopus laevis kommen Nervenzellen vor. Die intraependymalen Neurone sind rundlich und stehen mit dem Liquor cerebrospinalis durch eine breite Oberfläche in Berührung, von der sich längere und kürzere Fortsätze und ein Cilium (Typ 9+2) in das Lumen erheben. Die hypendymalen Neurone sind bipolar; ihr Dendrit verzweigt sich im Liquor ebenfalls in fingerförmige Fortsätze. Die Liquorkontaktfortsätze beider Zelltypen sind von feinen Filamenten ausgefüllt. Der Reissnersche Faden lagert sich manchen Fortsätzen an.In den intra- und hypendymalen Perikaryen findet man neben endoplasmatischem Retikulum, Golgi-Arealen und Mitochondrien kleine dense-core Vesikel (Durchmesser 600–900 Å). Der distale Fortsatz beider Neurontypen hat Neuritennatur. Axone, die synaptische und granulierte (Durchmesser 800–1200 Å) Vesikel enthalten, bilden relativ wenige Synapsen mit den Liquorkontaktneuronen. Im hypendymalen Neuropil findet man multipolare Nervenzellen, die 1000–1200 Å große granulierte Vesikel enthalten. Aufgrund des morphologischen Bildes wird die mögliche Rolle der Liquorkontaktfortsätze und des Ciliums bei der Funktion der Liquorkontaktneurone diskutiert.

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Ultrastructure of the spinal liquor contacting neurons in the clawed toad (Xenopus laevis)

Summary Nerve cells are situated between and below the ependymal cells of the central canal of the spinal cord of Xenopus laevis. The intraependymal neurons are round-shaped; they contact the cerebrospinal fluid by a large surface from which longer and shorter processes and a cilium (type 9+2) arise into the lumen. The hypendymal neurons are bipolar; their dendrite ramifies also into finger-like processes in the cerebrospinal fluid. The liquor contacting processes of both cell types contain fine filaments. The Reissner's fibre contacts some of the processes.In the intra- and hypendymal perikarya, small dense-core vesicles (diameter 600–900 Å) are found besides of endoplasmic reticulum, Golgi-areas and mitochondria. The distal process of both neuron types has neurite character. Axons containing synaptic and granulated (diameter 800–1200 Å) vesicles, form relatively few synapses with the liquor contacting neurons. In the hypendymal neuropile, multipolar nerve cells occur that contain granulated vesicles with a diameter of about 1000–1200 Å. On the basis of the morphological picture, the possible role of the liquor contacting processes and of the cilium in the function of the liquor contacting neurons are discussed.

     

Die Differenzierungsgenese der TSH-Zellen von Xenopus laevis unter Normalbedingungen und nach Thiouracilbehandlung

Zusammenfassung Die Strukturveränderungen der TSH-Zellen von Xenopus laevis im Verlaufe der Normalentwicklung und nach Belastung mit Methylthiouracil sind elektronenmikroskopisch untersucht worden. Folgende Ergebnisse wurden erzielt: Die thyreotropen Zellen lassen sich aufgrund ihrer polymorphen Granulation erstmals bei jungen Larven vereinzelt im Stadium 45, zahlreicher im Stadium 47, nachweisen. Form und Größe der sekretorischen Granula zeigen in verschiedenen Entwicklungs- und Funktionsphasen auffällige Veränderungen. Nach einem Durchmesserminimum im Stadium 49 K (frühe Ausschüttungsphase) erfolget eine Granulavergrößerung und Abrundung der Granulaform bis Stadium 59 K (Speicherphase). Bis Stadium 61 K ist eine Granulaverkleinerung und das Auftreten einer neuen, kleinen, stärker polymorphen Granulapopulation beobachtet worden (Ausschüttung und Neubildung).Nach MTU-Behandlung zeigt sich ein entsprechender Verlauf der Granulaveränderungen, jedoch mit höheren Durchschnittswerten und zeitlich wie stadienbezogenen Verschiebungen gegenüber den Kontrollen. Die funktionelle Deutung der Veränderungen des Granulastatus sowie die übrigen cytologischen Kriterien bestätigen und ergänzen unsere Vorstellungen vom Aktivitätsverlauf des Hypophysen-Schilddrüsen-Systems in der Amphibienontogenese.

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Differentiation of TSH-cells in Xenopus laevis under normal conditions and after thiouracil-treatment

Summary The ultrastructural changes of the TSH-cells in Xenopus laevis have been investigated in the course of normal development and after thiouracil-treatment. Individually at stage 45, and in larger numbers at stage 47, the thyrotropic cells are detected first in young larvae by their polymorph granulation. Significant changes in size and shape of these secretory granules occur during the different developmental and functional periods. The size-minimum at stage 49 K (early discharge period) is followed by an enlargement of the granules and a rounding-off of their shape up to stage 59 K (period of storage). Up to stage 61 K a decrease in granule-size as well as the appearance of a new population of small, more polymorph elements are to be observed (discharge and new formation).After MTU-treatment a corresponding course of granule-modifications is to be found, but if compared with the controls, there are larger granule-sizes as well as variations regarding age and stage of the animals. The granulation and other cytologic criteria confirm and complete current ideas of the functional development of the pituitary-thyroid-system during the ontogeny of the Amphibia.

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